Composition for Regenerating Hair Follicles Comprising CD36-Expressing Dermal Sheath Cells

ABSTRACT

Provided is a composition for regenerating hair follicles comprising CD36-expressing dermal sheath cells (DSc).

TECHNICAL FIELD

The present invention relates a composition comprising dermal sheath(DS) cells expressing CD36 antigen (thrombospondin receptor) (to bereferred to as “CD36-expressing DSc”) and arbitrarily, dermal papilla(DP) cells (to be referred to as “DPc”), a method for regenerating hairfollicles using such a composition, and an animal or three-dimensionalskin model having hair

BACKGROUND ART

Hair is viewed to be extremely important in terms of aestheticappearance. Thus, hair loss caused by congenital or acquired factors isa serious problem for many people. In today's society having a largeelderly population and containing numerous sources of stress inparticular, there are a growing number of opportunities for being atrisk to the loss of scalp hair due to various acquired factors. In orderto deal with this situation, various attempts have been made to providesuperior hair tonics that more effectively demonstrate hair growtheffects including the promotion of hair growth and thicker hair.

Hair follicles are exceptional organs that continue to repeatedlyself-regenerate throughout nearly the entire life of a mature livingorganism. Elucidation of the mechanisms of this self-regeneration areexpected to lead to clinical applications for which there areconsiderable demand, such as hair loss treatment using tissue celltransplants, and the construction of natural and functionally superiorskin sheets containing hair follicles and sebaceous glands. In recentyears, research has progressed rapidly on follicular epithelial stemcells (epithelial cells) accompanying the growing interest in stem cellresearch, and the properties of dermal papilla cells, which arefollicle-specific mesenchymal cells, have also been graduallydetermined. Dermal papilla cells fulfill the role of a so-called controltower that induces activation signals to follicular epithelial stemcells for self-regeneration of hair follicles, and have been determined,along with follicular epithelial stem cells, to be essential cells infollicle reconstruction evaluation systems (Kishimoto, et al., Proc.Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341: Non-Patent Document1).

Dermal papilla (DP) and dermal sheath (DS) surrounding the periphery ofhair follicles both differ from epithelial cells composing the majorityof hair follicles in that they are composed of mesenchymal-derived cellpopulations. With respect to DS, numerous findings have been reported inrecent years suggesting its importance with respect to hair follicleformation. DS has been reported to her regenerated from DP in anexperiment involving transplantation of hair bulbs—removed hairfollicles of rat whiskers (1), and follicle regeneration has beenreported to be induced in mice by transplanting DS of hair folliclesfrom which the lower half has been severed (2). In addition, Jahoda, etal. (Development, 1992, April:114(4), pp 887-897: Non-Patent Document 2)reported that follicle regeneration. can he induced by transplanting DSto humans (Horne, K. A. and Jahoda, C. A., Development, 1992,November:116(3), pp. 563-571: Non-Patent Document 3). Moreover, theTobin, Paus et al. group reported that cells migration occur between DSand DP, and that proliferation of dermal sheath cells (DSc) begins priorto DPc that begin to proliferated during the hair growth phase (Tobin,D. J. et al., J. Invest. Dermatol., 120, pp. 895-904, 2003: Non-PatentDocument 4).

In this manner, although DS has a high possibility of fulfilling animportant role in the formation of hair follicles, the mechanisms ofaction have not been well known, and even the properties of DSc are notunderstood. Therefore, we investigated the gene expression profile thatcharacterizes DSc, and analyzed the properties for the purpose ofclarifying the mechanism of action in the follicle formation.

PRIOR ARTS Non-Patent Documents

Non-Patent Document 1: Kishimoto, et. al Proc. Natl. Acad. Sci. USA(1999), Vol. 96, pp. 7336-7341

Non-Patent Document 2: Jahoda, et. al., Development, 1992, April:114(4),pp, 887-897

Non-Patent Document 3: Horne, K. A. and Jahoda, Development, 1992,November:116(3), pp. 563-571

Non-Patent Document 4: Tobin, P. S. et al., J. Invest. Dermatol., 120,pp. 895-904, 2003

Non-Patent Document 5: Linder, S. at al., Federation of AmericanSocieties for Experimental Biology, 14(2), 319 (2000)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novel follicleregeneration system.

Means for Solving the Problems

As a result of investigating the gene expression. profile of DSc using amicroarray, 304 genes were identified as genes with two or more higherexpression rate in DSc in comparison with that in DPc and fibroblasts(FBc). As a result of categorizing these genes by function usingGeneSpring's GeneOntology, many of the genes belonged to the category ofvascular-related factors, thereby suggesting involvement between DS andblood vessels. Among these, DSc were found to highly express CD36, andthis expression of CD36 by DSc was determined to be correlated withexpression of HGF (hepatocyte growth factor) that demonstrates hairgrowth promotional effects (J. Linder, et. al., Federation of AmericanSocieties for Experimental Biology, 14(2), 319 (2000) Non-PatentDocument 5).

Thus, the present application includes the inventions indicated below.

[1] A composition for regenerating heir comprising CD36-expressingdermal sheath (DS) cells.

[2] The composition for regenerating hair follicles of [1], furthercomprising dermal papilla (DP) cells.

[3] The composition of [2], wherein the ratio of the number ofCD36-expressing DSc to the number of DPc is about 10:1 to 1:10.

[4] The composition of [2] or [3], wherein the CD36-expressing DSc andthe DPc are both derived from mice or both derived from rats.

[5] The composition of [2] or [3], wherein the CD36-expressing DSc andthe DPc are heterologous cells, and each of them are derived from mice,rats or humans.

[6] A method for regenerating hair follicles, comprising transplantingthe composition of any of [1] to [5] to a human.

[7] A method for regenerating hair follicles by transplanting thecomposition of any of [1] to [5] to a recipient animal.

[8] The method of [7], wherein the recipient animal is animmunosuppressed animal.

[9] The method, of [7] or [8], wherein the recipient animal is animmunosuppressed animal selected, from the group consisting of a nudemouse, SCID mouse and nude rat.

[10] A method for regenerating hair follicles, comprising producing athree-dimensional skin model containing the composition of any of [2] to[5].

[11] A chimeric animal imparted with regenerated hair follicles bytransplanting the composition of any of [1] to [5] to a recipientanimal.

[12] The chimeric animal of [11], wherein the recipient animal is animmunosuppressed animal.

[13] The chimeric animal of [11] or [12], wherein the recipient animalis an immunosuppressed animal selected from the group consisting of anude mouse, SCID mouse and nude rat.

[14] A three-dimensional skin model imparted with regenerated hairfollicles by producing a three-dimensional skin model containing thecomposition of any of [2] to [5].

Effects of the Invention

The hair follicle regeneration composition of the present invention canbe used in transplant surgery regenerating hair follicles and inresearch and development on hair follicle reconstruction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the structure of it follicletissue.

FIG. 2 indicates expression level of various types of vascular-relatedfactors in various cells.

FIG. 3 is an image obtained by CD36 and CD31 immunohistochemicalstaining.

FIG. 4 is an image of whole mount stained hair. follicles obtained byCD36 and CD31 immunostaining.

FIG. 5 indicates the results of co-culturing CD36-positive DSc andvascular endothelial cells.

FIG. 6 indicates the results of HGF expression level in CD36-positiveDSc.

EMBODIMENTS TO CARRY OUT THE INVENTION

The present invention provides a composition for regenerating hairfollicles comprising DSc and arbitrarily comprising DPc, a method forregenerating hair follicles using such a composition, and an animal orthree-dimensional skin model having hair follicles regenerated by such amethod.

CD36 antigen is also referred to as thrombospondin receptor. CD36 is anintegral membrane protein found on the surfaces of numerous cell typesof vertebra animals, and is also known au FAT, SCARB3, GP88,glycoprotein IV (gpIV) and glycoprotein IIIb (gpIIIb). CD36 is a memberof the class B scavenger receptor family of cell surface proteins. Inaddition to thrombospondin, CD36 also binds with numerous other ligandssuch as collagen, erythrocytes parasitized by falciparum malariaparasites, oxidized low density lipoproteins, naturally-occurringlipoproteins, oxidized phospholipids and long-chain fatty acids. Inrecent research using genetically modified rodents, CD36 was able to beconfirmed to fulfill a definite role in fatty acid and sugar metabolism,heart disease, sense of taste and the transport of vegetable fats andoils in the intestinal tract. CD36 can also be involved in impairedglucose tolerance, athersclerosis, arterial hypertension, diabetes,cardiomyopathy and Alzheimer's disease.

Furthermore, the relation p between CD36 antigen and hair growth iscompletely unknown.

DSc are cells that compose the sheath portion that surrounds theperiphery of DP in hair follicles, and are mesenchymal cells in the samemanner as DPc. DP are considered to be derived from DS, and since DSproliferates prior to the proliferation of DP during the hair growthphase, DS is thought to supply DPc (Tobin, D. J. et al., J. Invest.Dermatol., 120, pp. 895-904, 2003: Non-Patent Document. 4).

Although there are no particular limitations thereon, DSc that expressCD36 can be sorted from DPc and other cells by, for example, commonlyused cell sorting techniques using antibodies, and preferably monoclonalantibodies, to CD36.

The DSc of the present invention can be derived from the skin of allmammals, such as humans, chimpanzees, other primates, domestic animalssuch as dogs, cats, rabbits, horses, sheep, goats, cows or pigs, andexperimental animals such as rats, mice or guinea pigs, and preferablynude mice, SCID mice or nude rats. In addition, the epidermal site maybe a site where there is hair growth such as the scalp, or a site wherethere is no hair growth such as the foreskin.

DPc (dermal papilla cells) refer to cells that are mesenchymal cellslocated in the lowest portion of hair follicles, and fulfill the role ofa so-called control tower by transducing activation signals tofollicular epithelial stem cells for self-regeneration of hairfollicles. Dermal papilla cell preparations containing only activateddermal papilla cells can be prepared according to for example, themethod described in Kishimoto, et al., Proc. Natl. Acad. Sci. USA(1999), Vol. 96, pp. 7336-7341 using transgenic mice. However, inconsideration of yield, and the like, these preparations are preferablyprepared by, for example, preparing a cell suspension by treating adermal tissue fraction, obtained by removing epidermal tissue from skintissue, with collagenase, followed by destroying the follicularepithelial cells by subjecting the cell suspension to cryopreservation.

The above-mentioned cryopreservation method can be specifically carriedout, for example, in the manner indicated below.

1. Mammalian skin is acquired.

2. The skin is then allowed to stand undisturbed for a suitable amountof time, such as overnight, in a protease solution such as a trypsinsolution as necessary, the epidermal portion is subsequently removedwith a forceps and the like, and the remaining dermis is treated with acollagenase to prepare a cell suspension.

3. The cell suspension is then filtered with a cell strainer asnecessary, and the sediment is removed by allowing to stand undisturbed.

4. After measuring the number of cells, the cells are re-suspended in acryopreservation solution at a suitable cell density such as 1×10⁵/ml to1×10⁸/ml, and the suspension is dispensed into small aliquots asnecessary and then placed in cryopreservation in accordance withordinary cell storage methods.

5. The cells are stored for a suitable amount of time and used afterthawing.

Although there are no particular limitations thereon, the freezingmethod consists of storing at a temperature of −20°C. or lower,preferably −50°C. or lower and more preferably −80° C. or lower in anultra-deep freezer, or in liquid nitrogen. Although there are noparticular limitations on the duration of cryopreservation, it is aperiod of, for example, 1 day or more, preferably 3 days or more andmore preferably 1 week or more to ensure that the epithelial cells arekilled. Furthermore, dermal papilla cells have been confirmed to remainviable even after storing for 4 months in liquid nitrogen. An ordinarycryopreservation solution used to store cells, such as CellBanker 2cryopreservation solution (Catalog No. BLC -2, Nippon Zenyaku Kogyo Co.,Ltd.), can be used for the cryopreservation solution.

Measurement of the number of cells can be carried out by a methodcommonly known among persons with ordinary skill in the art. Forexample, the number of cells can be measured by placing a cellsuspension obtained by diluting the cells with an equal volume of 0.4%Trypan blue stain (No. 15250-061, Invitrogen Corp.) on a hemocytometer(Eosinophil Counter, SLGC Co., Ltd.), and calculating the number ofcells in accordance with the method described in the instruction manualprovided with the hemocytometer.

Similar to DSc, the DPc of the present invention can be derived from theskin of all mammals, such as humans, chimpanzees, other primates,domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cowsor pigs, and experimental animals such as rats, mice or guinea pigs, andpreferably nude mice, SCID mice or nude, rats.

Preferably, the composition for regenerating hair follicles of thepresent invention further comprises epithelial cells. Epithelial cellsare cells that compose the majority of the dermis or epidermis of skin,and arise from a single layer of basal cells in contact with the dermisin using the example of mice, although epithelial cells derived fromnewborn mice (or fetuses) can be preferably used as epithelial cells,they may also be cells derived from mature skin, such as the epitheliumof resting phase hair or epithelium of growth stage hair, or culturedcells in the form of keratinocytes. These cells can be prepared from theskin of a desired donor animal according to methods commonly known amongpersons with ordinary skill in the art.

In a preferable aspect thereof, the epithelial cells can be prepared inthe manner described below.

1. Mammalian skin is acquired.

2. The epidermis of this skin is treated with trypsin by allowing tostand undisturbed overnight at 4° C.. in PBS containing 0.25% trypsin asnecessary.

3. After peeling off only the epidermal portion with a forceps and thelike and slicing into thin sections, the epidermal tissue is treated bysuspending for about 1 hour at 4° C. in a suitable culture broth (suchas keratinocyte culture broth).

4. The suspension is passed through a cell strainer having a suitablepore size, followed by subjecting to centrifugal separation andrecovering the epithelial cells.

5. The cell preparation is suspended in KGM or SFM medium at a desiredcell density, and then allowed to stand undisturbed on ice until justbefore the time of use.

Similar to DSc and DPc, the epithelial cells of the present inventioncan be derived from the skin of all mammals, such as humans,chimpanzees, other primates, domestic animals such as dogs, cats,rabbits, horses, sheep, goats, cows or pigs, and experimental animalssuch as rats, mice or guinea pigs, and preferably nude mice, SCID miceor nude rats. In addition, the epidermal site may be a site where thereis hair growth such as the scalp, or a site where there is no hairgrowth such as the foreskin.

Although there are no particular limitations on the ratio of DSc to DPcused, in the composition of the present invention, DSc and DPc arecontained at a ratio of preferably 1:10 to 10:1, and more preferably 1:3_(to) 3:1. Moreover, epithelial cells are contained at a ratio to thetotal number of DSc and DPc of 1:10 to 10:1, preferably 1:1 to 10:1,more preferably 1:1 to 3:1 and most preferably 1:1.

The combination of DSc, DPc and arbitrarily epithelial cells may be fromthe same species or different species. Thus, the composition forregenerating hair follicles of the present invention may be, forexample, a combination in which all of the DSc, DPc and epithelial cellsare derived from a human, a combination in which all of the DSc, DPc andepithelial cells are derived from the same species of mammal other thanhuman (the above combinations are combinations of the same species), acombination in which the DSc and DPc are derived from a human while theepithelial cells are derived a mammal other than human, a combination inwhich one of either the DSc or DPc are derived from a human, and theother and the epithelial cells are derived from the same species ordifferent species of mammal other than a human, and a combination inwhich one of either the DSc or DPc are derived a mammal other than ahuman and the other and the epithelial cells are derived from a human(the above combinations are combinations different species).

The method used to transplant the composition for regenerating hairfollicles of the present invention into a recipient animal can be inaccordance with a known transplant method. Reference can be made to, forexample, Weinberg, et al., Invest. Dermatol., Vol. 100 (1993), pp.229-236. For example, in the case of transplanting 1 nude mice, thecells that have been acquired are mixed just before or within 1 hourbefore transplant, the culture broth is removed by centrifugation(9000×g, 10 min), and after forming a cell aggregate of about 50 μL to100 μL, the cell aggregate is promptly poured into a siliconedome-shaped chamber embedded in the skin on the backs of the nude mice.Two weeks later, the chamber is carefully removed and starting anadditional 3 weeks later, the presence of hair formation at thetransplant site can be observed macroscopically. Transplantation for thepurpose of growing hair in animals including humans can he carried outby a similar method, and an appropriate method may be suitablydetermined by a physician or veterinarian.

In the case of transplanting the aforementioned composition into arecipient animal, that transplantation may be a homotransplantation,namely an autotransplantation, isotransplantation orallotransplantation, or may be a heterotransplantation. In the case of ahomotransplantation, the dermal papilla cell preparation and theepithelial cells are from the same species as the recipient. In the caseof a heterotransplantation, either the dermal papilla cell preparationor the epithelial cells are from a different species than the recipientwhile the other may be from the same species as the redolent, or bothmay be from a different species than the recipient. Examples ofrecipient animals include all mammals, such as humans, chimpanzees,other primates, domestic animals such as dogs, cats, rabbits, horses,sheep, goats, cows or pigs, and experimental animals such as rats, miceor guinea pigs, and preferably nude mice, SCID mice or nude rats.

In addition, a chimeric animal having regenerated hair follicles can beprovided by transplanting the aforementioned composition according tothe present invention into a suitable recipient animal. The resultingchimeric animal can serve as a useful animal model for, for example,researching and elucidating the mechanism of hair follicle regeneration,or screening drugs or herbs effective for regenerating hair follicles,growing hair or preventing hair loss. The recipient animal is preferablyan immunosuppressed animal regardless of the source of each of the cellscontained in system transplanted into the recipient animal. In addition,an animal species capable of being used as an experimental animal can beused for the animal species, and although any animal species may be usedprovided it coincides with the object of the present invention,preferable examples thereof include mice and rats. Among these animals,examples of immunosuppressed animals when using the example of miceinclude those in having the trait of a missing thymus in the manner ofnude mice. Furthermore, in consideration of the object of the presentinvention, particularly preferable examples of recipient animals includecommercially available nude mice (such as Balb-c nu/nu strain), SCIDmice (such as Balb/c-SCID), and nude rats (such as F344/N Jcl-run)

Moreover, by incorporating the composition according to the presentinvention in a three-dimensional skin model, a three-dimensional skin,model can be provided that has regenerated hair follicles. In this case,however, dermal papilla cells serving as a control tower. for hairgrowth are essential. A three-dimensional skin model can be produced inthe manner described below, for example, according to a method commonlyknown among persons with ordinary skill in the art (Amano, S. at al.,Exp. Cell Res. (2001), Vol. 271, no. 249-362). The tree -dimensionalskin model respectively contains DSc and DPc at 1×10⁶ cells/cm² to 1×10⁸cells/cm², preferably at 1.0×10⁷ cells/cm² to 1.5×10⁷ cells/m², and morepreferably at about 1.0×10⁷ cells/cm².

Method for Producing Three-Dimensional Skin Model

A suitable number of human fibroblasts are dispersed in DMEM containing0.1% collagen and 10% FBS followed by dispensing into a Petri dish andimmediately allowing to stand undisturbed in a CO₂ incubator at 37° C.After the medium has gelled, the gel is scraped from the side walls andbottom of the Petri dish so as to suspend the gel in the Petri dish. Thecells are then cultured while shaking the collagen gel to contract thegel to about one-fifth its original size and obtain a dermal model. Thedermal model is then placed on a stainless steel grid, a glass ring isplaced thereon, and 0.4 ml of cultured human epithelial cells (1.0×10⁶cells/ml) dispersed in KGM (keratinocyte growth medium) are injectedinto the ring and cultured. At this time, DSc and DPc are simultaneouslymixed and injected. Mouse neonatal epithelial cells can also be usedinstead of cultured human epithelial cells.

Medium consisting of DMEM, KGM, 5% FBS and Ca²⁺ is placed in the Petridish to a degree that the upper portion of the dermal model is exposedto air followed by culturing, and after about one week, the skin modelis observed and assessed for the presence or absence of rudimentary hairfollicle formation and regeneration.

Similar to the aforementioned chimeric animal having regenerated hairfollicles, a three-dimensional skin model having regenerated hairfollicles can be used for researching and elucidating the mechanism ofhair follicle regeneration, or screening drugs or herbs effective forgrowing hair or preventing hair loss.

The following provides a more detailed explanation of the presentinvention by indicating examples thereof.

Example 1

(Method)

Cell isolation and Culturing

Removal of the dermal portion of human scalp tissue. was carried outwith a scalpel in DMEM containing 10% fetal bovine serum.(Gibco/Invitrogen Corp.), and hair follicles were extracted from thesevered surface. Hair shafts containing outer root sheath cells (ORS,follicular epithelial cells) were removed from the hair follicles usingmicro-forceps so as to extract DP and DS. The isolated DP warestatic-cultured in a 35 mm collagen-coated tissue dish containingMedium-1 (Nissui low-serum fibroblast medium containing 10% fetal bovineserum, 10 ng/ml EGF, 20 ng/ml bFGF, 0.00075% β-mercaptoetbanol, 100units/ml (titer) penicillin, 0.1 mg/ml (titer) streptomycin and 0.25μg/ml (titer) amphotericin B), while the isolated DS were treated withcollagenase for 40 minutes at 37° C. followed by similarly static-culturing in a 35 mm collagen-coated tissue dish. After confirming thegrowth of both DP and DS one week later, the resulting DPc and DSc wereused as experimental samples. Commercially available cells (Toyobo Co.,Ltd.) were used for the fibroblasts (FBc). The DSc, DPc and FBc werestatic-cultured for 7 to 10 days using Medium-1. Subsequently, the cellswere subcultured using trypsin. Culturing conditions consisted ofculturing in a collagen-coated flask (T-75, Iwaki Glass Co., Ltd.) at37° C. and 5% CO₂. En addition, each of the cells used in the experimenthad been subcultured one to three times.

Normal human adult dermal microvascular endothelial cells (HMVEC, KuraboIndustries Ltd.) were used as vascular endothelial cells, and the cellswere cultured in low-serum growth medium (Humedia-MvG, Kurabo IndustriesLtd.) and then subcultured five times prior to use in the experiment.

Comparison of Gene Expression Profiles of DSc, DPc and FBc Using theMicroarray Method

Total RNA containing mRNA was collected from the DSc, DPc and FBc usingthe RNeasy Micro Kit (Qiagen Corp.). The collected RNA was subjected todouble-stranded cDNA synthesis using Agilent's protocol followed bysynthesis of cRNA labeled with cyanine 3.5. The labeled cRNA washybridized for 17 hours at 65° C. on a microarray chip slide (Agilent,Whole Human Genome (4×4.4K), G4110) since a two-color assay. Comparisonsof the gene expression levels of two types of cells each consisting ofDSc and DPc, DPc and FBc and FBc and DSc were carried out on each chipslide using two types each of RNA derived from the DS of two individuals(total of 4 types), two types each of RNA derived from the DP of twoindividuals (total of 4 types), and two types each of RNA derived fromthe FB of one individual. After washing the slides, images offluorescent signals (cyanine 3.5) of the cDNA on the chip were obtainedwith a dual-laser microarray scanner (Agilent Technologies Inc.). Theimage data was quantified using Feature Extraction Software Ver. 9.1,and at that time, taggings were made (tagged) to indicate abnormalvalues and low values at about the same level as background noisefollowed by analyzing the data. Each expression level was compared bycomparing two sets of quantitative values of the acquired signals.

(Analysis of Microarray Data)

Gene Spring GX 7.3.1 software (Agilent Technologies Inc.) was used toanalyze each gene expression level in greater detail usingbioinformatics techniques. Abnormal values and low values at about thesame level as background noise were tagged in a procedure using FeatureExtraction Software Ver. 9.1, and analyses were conducted using thosegenes that were not tagged. The genes for which there was a differencein expression level between two types of cells were extracted andsubjected to functional categorization by using GeneOntology(http://www.geneontology.org/). At that time, the degree of statisticalsignificance was tested using Fisher's exact test.

Cell Staining

Cell staining using CD36 antibody consisted of seeding DSc in a fourwell-chamber slide (Nalgene, Nalge Nunc International Corp.) subjectedto collagen surface treatment using an acidic collagen solution (KokenCo., Ltd.) and using the cells after culturing for 1 to 2 days. Afterwashing with PBS, the cells were fixed for 30 minutes with 4% PFA,washed with PBS and treated for 10 minutes with PBS solution containing0.1% Triton. X-100. After blocking for 30 minutes with PBS containing 3%BSA, the cells were allowed to react for 1 hour with a primary antibodysolution obtained by diluting CD36 antibody (ab17044, Abcam Inc.)50-fold with PBS containing 1% BSA. After washing for times with PBS,the cells were allowed to react for 1 hour with a secondary antibodysolution obtained by diluting Alexa 594-labeled anti-mouse IgG antibody(Invitrogen Corp.) 200-fold with PBS containing 1% BSA. After reactingwith DAPI solution in order to carry out nuclear staining, the cellswere washed four times with PBS and sealed with an anti-fade reagent(Prolong Gold Antifade Reagent, Invitrogen Corp.) and a cover glass. Thecells were observed using a fluorescence microscope (Olympus Corp.).

Tissue Staining

Human scalp tissue was embedded in a frozen tissue embedding agent (OTCCompound, Sakura Finetek Japan Co., Ltd.), and frozen section slideswere prepared with a frozen section production system (Cryostat, LeicaCamera AG). After fixing for 15 minutes with 4% PFA, the tissue waswashed with PBS and allowed to react for 1 hour using a blockingsolution obtained by adding 5% skim milk, 1% donkey serum and 0.1%Triton X-100 to PBS. Next, the tissue was allowed to react for 1 hour atroom temperature or overnight at 4° C. using primary antibody solutionobtained by diluting CD36 antibody solution (ab17044, Abcam Inc.) orCD31 antibody solution. (AF806, R&D Co., Ltd.) 50-fold and 100-fold,respectively, with the blocking solution. Furthermore, CD31 antibody wasused to label CD31 used as a vascular endothelial cell marker. Afterwashing 3 time with PBS, the tissue was allowed to react for 1 hour atroom temperature using a secondary antibody solution obtained bydiluting Alexa 594-labeled anti-mouse IgG antibody (Invitrogen Corp.) orAlexa 488-labeled anti-rabbit IgG antibody (Invitrogen Corp.) 200-foldeach with blocking solution. After reacting with DAPI solution, thetissue was washed 3 times with PBS and sealed with an anti-fade reagent(Prolong Gold Antifade Reagent) and a cover glass. The tissue wasobserved using a fluorescence microscope (Olympus Corp.).

Hair Follicle Whole-Mount Staining

Hair follicles isolated, from human tissue were fixed whileshake-culturing for 2 hours. at 4° C. with 4% PFA, The follicles weresequentially subjected to dehydration. treatment consisting of treatingfor 5 minutes each using 0.1% Tween PBS containing 25%, 50% or 75%ethanol to be referred to as PEST) and treating for 5 minutes each using100% ethanol. The treated samples were stored in ethanol at −20° C. Atthe time of use, after rehydrating with the same ethanol series PBST,the follicles were treated for 10 minutes with PS containing 5% TritonX-100. Subsequently, the follicles were sequentially reacted with theblocking solution used for tissue staining, a primary antibody solutioncontaining CD36 antibody. (ab17044, Abcam Inc.) and CD31 antibody (AP806R&D Co., Ltd.), a secondary antibody solution containing Alexa594-labeled anti-mouse IgG antibody and Alexa 488-labeled anti-rabbitIgG antibody, and DAPI solution. Furthermore, the follicles were washed.8 times each using PBS containing 0.1% Triton X-100 both between theantibody reaction procedures and after staining. Reaction conditionsconsisted of reacting overnight at 4° C. in the case of the primaryantibody solution, and reacting for 2 to 3 hours at 4° C. In the case ofthe secondary antibody solution. The follicles were observed with afluorescence microscope (Olympus Corp.) after sealing with an anti-fadereagent (Prolong Gold Antifade Reagent) and a cover glass.

RT-PCR

RNA was collected from the cells using TRIzol (Invitrogen Corp.) inaccordance with the protocol provided. The concentration of thecollected RNA. was measured with a nucleic acid quantification system(Nanodrop, Thermo Scientific Inc.). After making the concentrations ofRNA targeted for comparison to a same level, cDNA was synthesized usingoligo(dT) primers from the RNA using reverse transcriptase (SuperscriptIII, Invitrogen Corp.) in accordance with the Invitrogen protocol.Quantitative RT-PCR was then carried out using the synthesized cDNA astemplate and using LightCycler® FastStart DNA MasterPLUS SYPR Green(Roche Diagnostics GmbH) for the reaction reagent and LightCycler (RocheDiagnostics GmBH) for the reaction device. Composition conditions werein accordance with the Roche protocol. PCR conditions consisted ofinitial denaturation for 10 minutes at 95° C., denaturation for 10seconds at 95° C., annealing for 10 seconds at 60° C., and elongationfor 10 seconds at 72°C. The primer data used is as indicated below.

G3PDH: Forward primer: (SEQ ID NO: 1) 5′-GCACCGTCAAGGCTGAGAAC-3′Reverse primer: (SEQ ID NO: 2) 5′-ATGGTGGTGAAGACGCCAGT-3′ CD36:Forward primer: (SEQ ID NO: 3) 5′-GAGGAACTATATTGTGCCTATTCTTTGGC-3′Reverse primer: (SEQ ID NO: 4) 5′-CATAAAAGCAACAAACATCACCACACCAAC-3′CD31: Forward primer: (SEQ ID NO: 5) 5′-ATGCCGTGGAAAGCAGATACTCTAG-3′Reverse primer: (SEQ ID NO: 6) 5′-AATTGCTGTGTTCTGTGGGAGCAG-3′ HGF:Forward primer: (SEQ ID NO: 7) 5′-GAGGGAAGGTGACTCTGAATGAG-3′Reverse primer: (SEQ ID NO: 8) 5′-AATACCAGGACGATTTGGAATGGCAC-3′

The expression levels of each gene were measured using the softwareprovided. Furthermore, G3PDH was used as an internal standard, and thiswas used to correct the amount of cDNA of a control group whenquantifying each gene.

Cell Sorting

The cells were sorted using the Cell Separation Magnet (BD BiosciencesInc.). Operating conditions were in accordance with the protocolprovided by BD Biosciences Inc. After separating the cells using trypsinsolution, the cell suspension was passed through a cell strainer havinga pore size of 70 μm (Falcon Inc.) followed by counting the number ofcells. 5 million to 10 million of cells were suspended in 500 ml of PBSsolution containing 3% fetal bovine serum, followed by the addition ofCD36 antibody (ab17044, Abcam Inc. so as to be diluted 50-fold andallowing to react for about 15 minutes on ice. After recovering thecells by washing and centrifuging using 1×Imag Buffer (BD BiosciencesInc.), the cells were re-suspended in 30 μl of anti-mouse IgG1 magneticparticles (BD Biosciences Inc.) and allowed to stand undisturbed for 30minutes on ice. 500 μl of 1×Imag Buffer (BD Biosciences Inc.) were addedfollowed by placing in the Cell Separation Magnet (BD Biosciences Inc.)and allowing to stand undisturbed for 8 minutes. The supernatant wasrecovered while being careful so as not to dislodge cells adhered to thelateral surfaces by the magnet, and the resulting supernatant was usedas CD36-negative DSc. Continuing, after again adding 500 μl of 1×ImagBuffer (BD Biosciences Inc.) and suspending cells adhered to the lateralsurfaces, the suspension was placed in the Cell Separation Magnet andallowed to stand undisturbed for 4 minutes followed by removal of thesupernatant. This procedure was further repeated once and the cellsadhered to the lateral surfaces were used as CD36-positive DSc. Therecovered CD36-positive and CD36-negative DSc were suspended in Medium-1followed by culturing for 2 to 4 days. at 37° C. and 5% CO₂ using acollagen-coated flask. (T-25, Iwaki Glass Co., Ltd.) for the culturevessel, followed by use in the experiment.

Co-Culturing Experiment

An experiment was conducted using the CD36-positive and CD-36 negativeDSc derived from each specimen for N=3 and 4 times, respectively,300,000 of the sorted CD36-positive and CD36-negative DSc wererespectively seeded to a collagen-coated flask (T-75). Subsequently,after culturing for 2 days in Medium-1, 400,000 HMVEC were added andco-cultured for 1 day in Humedica-MvG (Kurabo Industries Ltd.).Subsequently, the medium was replaced with medium obtained by adding 100units/ml (titer) of penicillin, 0.1 mg/ml (titer) of streptomycin, 0.25μg/ml (titer) of amphotericin B and 0.1% BSA (Sigma Corp.) to vascularendothelial cell basal medium (Humedia-EB2, Kurabo Industries Ltd.).After further co-culturing for 1 day, the cells were separated withtrypsin solution followed by proceeding with analysis using FACS. Thecells were then passed through a 70 μm cell strainer (Falcon Inc.),suspended in PBS solution containing 3% fetal bovine serum, and allowedto react for 20 minutes on ice using a primary antibody solution, i.e.,CD31 antibody solution (AF806, R&D Co., Ltd.). After washing the cellswith PBS solution containing 3% fetal bovine serum, the cells wereallowed to react for 20 minutes on ice with secondary antibody solution,i.e., Alexa 488-labeled anti-rabbit IgG antibody (Invitrogen Corp.) andre-suspended in 0.5 ml of PBS solution followed proceeding with analysisusing Cell Lab Quanta 5C (Beckman Coulter Inc.). Preparations, includinglaser accuracy management, were made using the protocol and workingreagent designated by Beckman Coulter. The number CD31-positive cellswas measured using the FL1 channel (525 nm). Furthermore, correction wasmade to eliminate autofluorescence by using endothelial cells that didnot react with CD31 antibody. Following measurement, the number ofCD31-positive cells was calculated based on total number of cellsobtained and the ratio of CD31 -positive cells.

(Results)

Table 1 shows the expression level of some vascular-related factors.Although vascular-related factors were determined to be highly expressedin DSs, CD36 and HGF were determined to be specifically highly expressedin DSs. FIG. 2 indicates the expression level of various types ofvascular-related factors in DSc, DPc, ORS cells and VEC (vascularendothelial cells). CD36 and HGF were determined to be expressedextremely specifically in DS. On the basis of cell staining results aswell, CD36-positive cells were observed to be present only in isolatedDS cultured cells, while CD36-positive cells were determined to beabsent in DPc or FBc (data not shown).

TABLE 1 Vascular-Related Factors Highly Expressed in DSc DS/DP DS/FBFB/DP CD36 40.24 10.97 3.93 HGF 20.19 3.28 10.34 14.52 4.33 4.45 17.854.14 4.47 TBX2 (T-box2 (transcription factor)) 20.67 4.68 6.07 VSGFA(vascular endothelial cell growth 3.41 5.12 0.50 factor) 4.34 7.40 0.482.22 4.16 0.52 2.68 4.55 0.60 FDGFA (platelet-derived growth factor)5.82 3.67 1.80 COX-1 (prostaglandin synthase) 11.73 2.24 4.88 9.64 1.605.83

Specific staining of CD36 was also observed in the dermal sheaths ofhair follicles, namely DS as a result of immunohistochemical staining ofCD36 and CD31 on the sections of hair follicle (FIG. 3). Moreover, theresults of hair follicle whole-mount staining indicated dense areas ofblood vessels in a portion of the DS, and CD36-positive DSc weredetermined to be localized in those dense areas. Thus, CD36-positive DScwas suggested to be intimately involved with blood vessels. In addition,although CD36-positive DSc cells are nearly always co-localized withblood vessels, CD36-positive DSc are absent in the vicinity of somevessels (FIG. 4).

Thus, CD36-positive DSc cells were suggested to promote vascularizationby, for example, promoting the proliferation of vascular endothelialcells.

In an experiment in which CD36-positive DSc isolated by cell sortingwere co-cultured with vascular endothelial cells, the CD36-positive DScwere indicated to significantly promote the proliferation of vascularendothelial cells in comparison with CD36-negative DSc (FIG. 5).Moreover, isolated CD36-positive DSc were also indicated to highlyexpress HGF in comparison with CD36-negative DSc cells (FIG. 6). Asmentioned above, HGF is commonly known as a factor that promotes thegrowth of new hair and hair growth (Non-Patent Document 5). Thus,transplantation of CD36-positive DSc into hair follicles is clearlyeffective for the growth of new hair and hair growth

1. A composition for regenerating hair follicles, comprisingCD36-expressing dermal sheath cells (DSc).
 2. The composition forregenerating hair follicles according to claim 1, further comprisingdermal papilla cells (DPc).
 3. The composition according to claim 2,wherein the ratio of the number of CD36 -expressing DSc to the number ofDPc is about 10:1 to 1:10.
 4. The composition according to claim 2 or 3,wherein the CD36 -expressing DSc and the DPc are both derived from miceor both derived from rats.
 5. The composition according to claim 2 or 3,wherein the CD36 expressing DSc and the DPc are heterologous cells, andeach of them are derived from mice, rats or humans.
 6. A method forregenerating hair follicles, comprising transplanting the compositionaccording to any of claims 1 to 5 to a human.
 7. A method. forregenerating hair follicles by transplanting the composition accordingto any of claims 1 to 5 to a recipient animal.
 8. The method accordingto claim 7, wherein the recipient animal is an immunosuppressed animal.9. The method according to claim 7 or 8, wherein the recipient animal isan immunosuppressed animal selected from the group consisting of a nudemouse, SCID mouse and nude rat.
 10. A method for regenerating hairfollicles, comprising producing a three-dimensional skin modelcontaining the composition according to any of claims 2 to
 5. 11. Achimeric animal imparted with regenerated. hair follicles bytransplanting the composition according to any of claims 1 to 5 to arecipient animal.
 12. The chimeric animal according to claim 11, whereinthe recipient animal is an immunosuppressed animal.
 13. The chimericanimal according to claim 11 or 12, wherein the recipient animal is animmunosuppressed animal selected from the group consisting of a nudemouse, SCID mouse and nude rat.
 14. A three-dimensional skin modelimparted with regenerated hair follicles by producing athree-dimensional skin model containing the composition, according toany of claims 2 to 5.